Semi-synthesis of human insulin

ABSTRACT

A process for preparing a B30-threonine-insulin which comprising reacting a des-B30-insulin with an excess amount of threonine derivative in the presence of an enzyme specifically acting on a lysine carbonyl in peptide bondings and produced by Achromobacter lyticus M 497-1.

This invention relates to a semi-synthesis of human insulin. Moreparticularly, it consists of an enzymatic synthesis of aB30-threonine-insulin including human insulin. The synthesis is effectedby reacting a des-B30-insulin with an excess amount of threoninederivative in the presence of an enzyme specifically acting on a lysinecarbonyl in peptide bondings which is produced by Achromobacter lyticusM 497-1 and removing any protecting group of the threonine to yield aB30-threonine insulin.

Insulin is an indispensable medicament for treatment of diabetes. Bovineand porcine insulins are commercially available at present. Theseinsulins are, however, different from human insulin in a few amino acidcomponents, which cause formation of antibody in patients. The antibodyis known to decrease the effectiveness of further insulin treatment.Therefore, human insulin is favoured and its commercial availability hasbeen eagerly desired. Human insulin is different from other animalinsulin in amino acid components at the positions 8, 9, and 10 of the Achain and at the position 30 of the B chain. This invention provides aprocess to substitute the amino acid at the 30 position of the B chainof an animal insulin with L-threonine. Accordingly, human insulin can beeasily prepared by the process of this invention using porcine insulinas starting material. Porcine insulin is different from human insulin inthe amino acid at the B30 position. Thus-obtained human insulin isfavourably used for treatment of diabetic patients. Further, thisinvention provides other B30-threonine-insulins starting from otheranimal insulins. The resultant insulins are different from human insulinin amino acids at 8, 9, and 10 positions of the A chain and can be usedas reagents to test antigenicity and as medicament for diabetes.

Human insulin was chemically prepared by M. A. Ruttenberg as shown inU.S. Pat. No. 3,903,068 and by R. Obermeier et al. as described inHoppe-Seyler's Z. Physiol. Chem., 357, 759-767 (1976). These processescomprise condensation of a desoctapeptide-(B23-30)-porcine insulin witha synthetic octapeptide corresponding to positions B23-30 of humaninsulin. The former process, however, includes alkaline hydrolysis whichis accompanied by adverse side-reactions. The latter process is also anon-specific reaction which brings about many side-reactions andrequires complicated purification procedures. Therefore, these processescannot be applied on an industrial scale.

M. Bodanszky et al. provide a process for preparing human insulin inU.S. Pat. No. 3,276,961 wherein human insulin was prepared from otheranimal insulins by an action of an enzyme such as carboxypeptidase A andtrypsin in the presence of threonine. This process is not likely toproduce human insulin because trypsin and carboxypeptidase A hydrolyzenot only the peptide bond of lysyl-alanine (B29-B30) but also the otherpositions in insulin under the condition described there. Trypsinpreferentially hydrolyzes the peptide bond of arginyl-glycine (B22-B23)rather than that of lysyl-alanine (B29-B30). Meanwhile, carboxypeptidaseA cannot release solely the alanine at the C-terminal of the B chainwithout liberating asparagine at the C-terminal of the A chain. Aspecial condition, i.e. reacting in an ammonium hydrogencarbonate buffersolution, is necessary to prevent the release of the asparagine. Thecondition was discovered in 1978 (Hoppe-Seyler's Z. Physiol. Chem., 359,799-802 (1978)). Furthermore, peptide synthesis may hardly occur becausethe hydrolysis ratio is faster than the synthesis ratio under thisspecial condition.

Therefore, it is concluded that there has been no industrially availableprocess for preparing human insulin until now.

This invention, however, can provide human insulin on a commercialscale. The process consists of enzymatic condensation of ades-B30-insulin with a L-threonine derivative. The process is differentfrom any known process and has many advantages. It is a specificreaction not accompanied by any adverse side reaction such asracemization. Additionally, unreacted starting materials can be easilyrecovered without damage and can be reutilized. The present process iseffected in the presence of an enzyme produced by Achromobacter lyticusM 497-1, which is known, specifically spliting a lysine carbonyl bondingin peptide and called protease I by Masaki et al. in Agric. Biol. Chem.42, 1443 (1978). The present inventors discovered that the enzyme has anactivity to make a peptide bond between the lysine carboxyl at theC-terminal of the B chain of des-B30-insulin and a L-threoninederivative. Furthermore, the process uses an excess amount ofL-threonine derivative. The excess amount of the threonine derivativeprevents the breakage of the carbonyl bond of the lysine at the 29position of the B chain, although the bond is naturally split by theabove enzyme.

The process comprises a reaction of a des-B30-insulin (noted as CompoundII hereinafter) with an excess amount of L-threonine derivative (notedas Compound I hereinafter) in the presence of an enzyme specificallyacting on a lysine carbonyl in peptide bondings and further subjectingthe product to deprotecting-group reaction to yield a B30-threonineinsulin.

Compound I is represented by the following formula:

    Thr(R.sup.1)(R.sup.2)

wherein Thr is an L-threonine residue; R¹ is hydrogen or ahydroxy-protecting group; and R² is a carboxyl-protecting group.

Compound II can be prepared by reacting various animal insulins (e.g.pig, cattle, whale, sheep, rabbit, fish, monkey and the like) withcarboxypeptidase A as disclosed by E. W. Schmitt et al. inHoppe-Seyler's Z. Physiol. Chem. 359, 799 (1978). It is also prepared byusing a protease produced by Achromobacter lyticus. The protease has aspecificity to lysine and specifically splits the lysine carbonyl bondin peptides as noted above. The isolation and characteristics aredescribed by Masaki et al. in Agric. Biol. Chem., 42, 1443 (1978). Thepreparation of des-B30-porcine insulin is disclosed in Biochem. Biophys.Res. Commun. 92, 396-402 (1980) and Japanese Patent Publication(Not-examined) No. 54-135789. Des-B30-insulins of other animals may beprepared in the same manner.

The hydroxy group of Compound I may be protected or not. Both theprotected and unprotected compounds can be subjected to the reaction.The protecting group to be used is selected from any hydroxy-protectinggroup generally used in peptide synthesis such as t-butyl, benzyl,acetyl and the like. The carboxyl group of Compound I has to beprotected and any carboxyl-protecting group utilized in peptidesynthesis can be employed. They are, for example, alkyl esters (e.g.t-butyl ester), aralkyl esters (e.g. benzyl ester), amide, substitutedamides (e.g. anilide), salts (e.g. sodium) and so forth.

Protecting groups should be chosen considering the influence of theintroduction and removal reactions to insulin because the reactionsmight result in denaturing or inactivation of insulin. If ahydroxy-protecting group and a carboxyl-protecting group of thethreonine residue are suitably selected, one removal procedure is enoughto remove both the protecting groups. Protecting groups used in peptidesynthesis are detailed by M. Bodanszky et al. in Peptide Synthesis, thesecond edition (1976) published by John Wiley & Sons.

The enzyme used in the present invention is an enzyme specificallyacting on lysine carbonyl in peptide bondings which is produced byAchromobacter lyticus M 497-1 and called Protease I as noted above. Theisolation and characteristics are disclosed in the above-mentionedreference.

The condensation of Compound I with Compound II is effected undersuitable conditions for the peptide synthesis of the above enzyme.

The reaction is preferably effected at about pH 5 to 9, more preferablyabout pH 6 to 7. Reaction temperature is about 0° to 50° C., preferablyabout 20° to 40° C.

It is desired that both concentrations of Compound I and Compound II areas high as possible. The preferable molar ratio of Compound I andCompound II is about 5:1 to 1000:1, especially about 25:1 to 200:1.Water-miscible organic solvents are preferably added to the reactionmixture. The addition of organic solvent lowers the aqueousconcentration of the reaction mixture resulting in prevention of reversereaction, i.e. hydrolysis of product, and also remarkably increases thesolubility of Compound I and Compound II. The organic solvents to beused are, for example, methanol, ethanol, dimethylformamide, dimethylsulfoxide, glycerol and the like. The preferred solvents are ethanol anddimethylformamide. The solvents can be used singly or as a mixture. Thepreferable concentration of the organic solvent is about 0 to 65%,especially about 40 to 60% of the reaction mixture. The ratio of theorganic solvent should be determined by the solubility of startingmaterials, decline of enzyme to denature and its hydrolyzing activity.

Reaction medium may be chosen from trishydroxymethylaminomethane(abbreviated tris hereinafter), carbonate, borate buffer solutions andthe like. Enzyme concentration is determined depending on concentrationof substrates and enzyme activity. For example, Protease I is usedpreferably in a concentration from about 0.01 mg/ml to 100 mg/ml. Enzymemay be used intact or in a fixed form such as a combination with or aninclusion in insoluble carrier such as cellulose, dextran (e.g. Sephadex(trade mark)), agarose (e.g. Sepharose (trade mark)), polyacrylamide gel(Bio-gel (trade mark)), porous glass and the like.

Reaction time is variable and is affected by other reaction conditions.Reaction may be continued until substrate and product reach equilibrium.It takes generally about 3 to 72 hours and in most cases about 6 to 24hours.

The product insulin may be isolated by combining the usual method usedin peptide chemistry. An isolation procedure is illustrated as follows:The reaction mixture is applied to gel filtration to isolate and collectunreacted Compound I and enzyme. The recovered Compound I and enzyme canbe reutilized. The remaining part is applied to suitable chromatographyto isolate a resultant protected-insulin and an unreacteddes-B30-insulin. The latter can be reused as Compound II. The former issubjected to reaction for removal of the protecting group.

The removal of protecting group is effected according to the usualmanner, though the method should be chosen depending on the propertiesof the protecting group. The t-butyl group is exemplified as aprotecting group for both the hydroxy and carboxyl group of threonine.It may be removed by treating with trifluoroacetic acid in the presenceof a cation-trapping agent, e.g. anisole. As noted above, if a soletreatment may remove the hydroxy-protecting and the carboxyl-protectinggroups at once, the process becomes simple and results in good yield.When the C-terminal of the resultant insulin is protected with otherester, amide, substituted amide or salt, the protecting group may beremoved by suitable hydrolysis or desalting technique.

The insulin prepared by this invention is useful for treatment ofdiabetes and also as a reagent.

The insulin obtained by this invention shows the same activity oflowering blood sugar level as bovine insulin in mice as follows:

Test Method: Test compound is dissolved in 0.01 M hydrochloric acid andthe solution is diluted to a suitable concentration (2.5-20 μg/ml) with20 to 60 times of physiological salt solution. The resultant solution isintravenously administered in a portion of 0.1 ml/10 g body weight to DSmice fasted for 5 hours. Blood is taken from orbital blood vessel 45minutes after the administration and blood sugar is measured byglucose-oxidase method using a commercial kit produced by BoehringerMannheim Corporation.

                  TABLE 1                                                         ______________________________________                                        Insulin Activity                                                              Test      Dose            Blood Glucose Level                                 Compound  (μ9/10 g Body Weight)                                                                      (mg/dl)                                             ______________________________________                                        Physiological                                                                 Salt Solution                                                                           --              112.2 ± 7.3 (1)                                  Semi-synthetic                                                                          0.25             80.2 ± 2.6 (2)                                  Human Insulin                                                                           1                41.0 ± 6.0 (3)                                  Bovine Insulin                                                                          0.25             73.2 ± 1.5 (4)                                            1                41.0 ± 2.3 (5)                                  ______________________________________                                         Note:                                                                         (2)-(4), (3)-(5) No significant difference                               

Human insulin prepared by this invention can be administered to humanbeings in the same manner as porcine or bovine insulin preparations onthe market. It may be made into pharmaceutical preparations in the usualmanner. For example, it may be made into an injectable solution by asuitable procedure such as those for preparing zinc complex with zincchloride, a buffered solution with a suitable buffer such as sodiumhydrogenphosphate, sodium acetate and the like, and an isotonic solutionwith sodium chloride. Furthermore, suitable antiseptics may be addedthereto. They are, for example, cresol, phenol, para-hydroxybenzoic acidalkyl esters (e.g. methyl, ethyl, propyl, butyl esters and the like).The dosage of the human insulin is the same as insulin preparations onthe market. Namely, about 1 to 100 units may be administered to a humanadult per day, though dosage depends on the seriousness of diabetes.

The following example is given to further illustrate and describe thepresent invention. Each abbreviation has the meaning as follows:

Ala: alanine, Arg: arginine, Asp: aspartic acid,

CysO₃ H: cysteic acid, Glu: glutamic acid, Gly: glycine,

His: histidine, Ile: isoleucine, Leu: leucine, Lys: lysine,

Phe: phenylalanine, Pro: proline, Ser: serine,

Thr: threonine, Tyr: tyrosine, Val: valine,

OBu^(t) : t-butyl ester residue.

EXAMPLE (1) Desalanine-(B30)-procine insulin

To a solution of procine insulin (500 mg) in 0.1 M ammoniumhydrogencarbonate (100 ml, pH 8.3) is added crystalline carboxypeptidaseA (5 mg, Worthington Co., pretreated with diisopropylfluorophosphate, 49u/mg). The mixture is incubated at room temperature for 8 hours. Theincubation is stopped when alanine is released at a ratio of 0.77 M/1 Minsulin. The reaction mixture is lyophilized. The product is dissolvedin 0.5 M acetic acid and adsorbed on a column of Sephadex G 50 (3.5×95cm) of super fine particles. The column is eluted with 0.5 M acetic acidwith a portion of 11.5 ml per one fraction. Fraction Nos. 40-60 arecollected and lyophilized to give the title compound (460 mg). Yield is92%.

The product is hydrolyzed for amino acid analysis with 6 M hydrochloricacid at 110° C. for 24 hours to give the following result. The figuresin the parenthesis show the theoretical values throughout thisspecification.

Amino acid analysis: Lys 1.00 (1), 1.91 (2), Arg 0.95 (1), Asp 3.21 (3),Thr 2.09 (2), Ser 2.97 (3), Glu 7.35 (7), Gly 4.29 (4), Ala 1.26 (1),CysO₃ H 5.94 (6), Val 3.86 (4), Ile 1.55 (2), Leu 6.53 (6), Tyr 4.08(4), Phe 3.22 (3), Pro 1.2 (1).

(2) [B30-Thr-OBu^(t) ]-porcine insulin

The above product (100 mg, 10 mM) and L-threonine t-butyl ester (205 mg,500 mM) are dissolved in a mixture (1.05 ml) of ethanol anddimethylformamide (1:1, v/v). The solution is mixed with a 0.5 M boratebuffer solution (0.7 ml) of Protease I (0.35 mg) produced byAchromobacter lyticus M 497-1 and incubated at 37° C. for 2 days. Thereaction mixture is acidified with glacial acetic acid and applied togel filtration with a column (4.2×130 cm) of Sephadex G 50 of super fineparticles to separate into an enzyme fraction, an insulin fraction and athreonine fraction. The enzyme and L-threonine t-butyl ester arerecovered in an amount of more than 50%. The insulin fraction islyophilized to give a crude powder (83 mg).

The product is applied at 4° C. to a column of DEAE-Sephadex A 25(1.9×24.5 cm) which is previously equilibrated with 0.01 M tris buffersolution (pH 7.6) and 7 M urea. The column is eluted with the abovebuffer solution (800 ml) and then a linear Na⁺ gradient (to 0.3 M NaCl)is performed to give a fraction A near to 0.08 0.09 M concentration anda fraction B near to 0.13 0.14 M. Each fraction is immediately dialyzedagainst 0.01 M ammonium acetate for 3 to 4 days in a cool place andlyophilized to give a powder (48 mg) from the fraction A and a powder(20 mg) from the fraction B. The former is identified to be the titlecompound and the latter is identified to be a mixture ofdesalanine-(B30)-porcine insulin and contaminated porcine insulin byhigh precious liquid chromatography and polyacrylamide gelelectrophoresis.

(3) Human insulin

Trifluoroacetic acid (2 ml) with anisole (0.2 ml) is added to theproduct (40 mg) obtained above and the mixture is kept at roomtemperature for 30 minutes. The trifluoroacetic acid is removed undernitrogen atmosphere and the mixture is extracted with ether (15 ml)after addition of 1 N acetic acid (2 ml) to remove the anisole. Theacetic acid layer is lyophilized to give the title compound (37 mg).Yield is 56% when calculated from the starting compound; 76% purity.

The product is identified as the title compound by amino acid analysis,Slab gel electrophoresis and high precious liquid chromatography. Aminoacid analysis is performed under the same condition as in (1) and theresult is as follows:

Amino acid analysis: Lys 1.00 (1), His 1.90 (2), Arg 0.93 (1), Asp 3.21(3), Thr 3.05 (3), Ser 2.99 (3), Glu 7.23 (7), Pro 1.25 (1), Gly 4.33(4), Ala 1.25 (1), Val 3.91 (4), Ile 1.58 (2), Leu 6.51 (6), Tyr 3.66(4), Phe 3.15 (3).

What is claimed is:
 1. A process for preparing a B30-threonine-insulinwhich comprises reacting a des-B30-insulin with a threonine derivativeof the formula

    Thr(R.sup.1)(R.sup.2)

wherein Thr is an L-threonine residue, R¹ is hydrogen or ahydroxy-protecting group and R² is a carboxyl-protecting group, in amolar ratio of the threonine derivative to the des-B30-insulin of 5:1 to1000:1, and in the presence of an enzyme specifically acting on thelysine carbonyl in peptide bondings and produced by Achromobacterlyticus M 497-1, and subjecting the resultant product to one or morereactions to remove all of the protecting groups.
 2. The processaccording to claim 1, wherein the molar ratio of the threoninederivative to the des-B30-insulin is 25:1 to 200:1.
 3. The processaccording to claim 1, wherein the reaction is effected in a mediumcontaining one or more water miscible organic solvents.
 4. The processaccording to claim 3, wherein the organic solvent is selected from thegroup consisting of methanol, ethanol, dimethylformamide, dimethylsulfoxide and glycerol.
 5. The process according to claim 4, wherein theorganic solvent is selected from the group consisting of ethanol anddimethylformamide.
 6. The process according to claim 1, wherein thedes-B30-insulin is des-B30-porcine-insulin.
 7. The process according toclaim 1, wherein the des-B30-insulin is des-B30-bovine-insulin.
 8. Theprocess according to claim 1, wherein the threonine derivative isL-threonine t-butyl ester.
 9. The process according to claim 1, whereinthe reaction is effected at pH 5 to pH
 9. 10. The process according toclaim 9, wherein the reaction is effected at pH 6 to pH
 7. 11. Theprocess according to claim 1, wherein the reaction is effected at atemperature of 0° to 50° C.
 12. The process according to claim 11,wherein the reaction is effected at a temperature of 20° to 40° C.